In 2009, an estimated 197,300 women with early stage invasive breast cancer and/or carcinoma in situ (CIS) received breast conserving surgery (BCS). BCS involves removal of malignant tissue with a surrounding margin of normal breast tissue. Residual cancer found in this margin after surgery is an important predictor of local recurrence of cancer after BCS. Recently, it was found that one death was averted for every four women in which a local recurrence is avoided. Thus, complete tumor excision is essential to reduce the risk of recurrence. As many as 20-70% of BCS patients must undergo re-excision surgery, because their cancer was incompletely removed during the first BCS procedure. This represents an enormous physical burden to the patient (increasing her chances for surgical complications and/or eventual cancer-related mortality) and financial burden to the health care system (effectively doubling the cost of treatment for this group of patients). By 2015, it is expected that the number of patients undergoing BCS will rise from approximately 197,000 to more than 270,000 per year in the U.S., at an annual growth rate of 5.5%. With no industry standard to prevent re-excision, it is expected that there will be a concomitant rise in the number of re-excision surgeries. Thus, there is a significant unmet clinical need for effective intra-operative assessment of breast tumor margins. The long-term research objective of this application is to develop a clinically-viable system for wide-field high-resolution microscopy of the tumor bed in BCS procedures, using a wide-field optically-sectioned scanning imager optimized for in vivo imaging, and non-toxic tissue-enhancing fluorescent contrast agents. This would be combined with automated image classification algorithms, which confer an automatic decision to the surgeon intra-operatively, whether or not additional tissue should be resected from the tumor bed. The objective of this development-phase R21 proposal is to complete a critical technology refinement, and to validate it in a pre-clinical tumor margin model. The specific aims of the current R21 proposal are 1) To develop a practical, non-contact, optically-sectioned wide-field fluorescence imaging system for in vivo tumor bed assessment, and 2) To evaluate the imaging system for in vivo tumor margin assessment in a transgenic murine tumor margin model. Successful completion of the R21 will result in a critical first step towards a clinically-viable system for in vivo fluorescence histology of breast tumor margins.